JP2013188181A - Method and reagent for detecting gonococcus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method that can quickly obtain a result with high sensitivity and high specificity compared with a conventional examination kit in a gonococcal examination.SOLUTION: A polynucleiotide including a specific base sequence is amplified and detected using a first primer comprising a specific base sequence having a sequence homologous to a part of a specific base sequence in a gonococcus 16S rRNA, and a second primer comprising a specific base sequence having a sequence complementary to a part of the specific base sequence.

Description

  The present invention relates to a method for detecting gonococci rapidly and with high sensitivity, and a detection reagent using the method. The present invention is useful in the fields of clinical examination and public health.

  Neisseria gonorrhoeae is a Gram-negative bacilli that belongs to the genus Neisseria and is a causative microorganism of Neisseria gonorrhoeae infection.

  Neisseria gonorrhoeae infection is a common infection among sexually transmitted diseases along with genital chlamydia infections. It is mainly transmitted through sexual activity, causing urethritis in men and cervicitis and urethritis in women. In severe cases, epididymis occurs in men and pelvic inflammatory disease in women.

  In recent years, reflecting the diversification of sexual behavior, cases of extragenital infections such as pharyngeal and rectal infections are increasing. In such a case, the symptoms are often poor, and may become a source of infection to others without consciousness (Non-Patent Document 1).

  In Japan, gonococcal infection is one of the fixed-point grasp diseases, and the trend survey is being conducted. According to it, in men, the number of gonorrhea infections is almost equal to the number of genital chlamydial infections, each accounting for about 40% of all sexually transmitted diseases. On the other hand, in women, gonococcal infection accounts for about 10% of all sexually transmitted diseases (Non-patent Document 2).

  As for gonococcal infection, in general, many women are asymptomatic even if they are infected (asymptomatic infection). Therefore, it is often the main source of infection for gonorrhea in men without treatment. In particular, in many cases where the gonococcal infection of the pharynx is a inflammatory symptom, the examination is often not performed.

  As described above, gonococcal infection of women and gonococcal infection of the throat often have relatively minor symptoms, and there are many patients who do not visit a medical institution. However, even asymptomatic patients can be a source of gonorrhea to others. In the case of women, if the infection is left untreated, serious complications such as ectopic pregnancy, infertility, and neonatal conjunctivitis due to birth canal infection during delivery may occur. Therefore, it is necessary to surely treat when an infection with gonococcus is found, and it is very important to perform an gonococcal test on patients and pregnant women suspected of infection.

Japanese Patent No. 3241717 JP 2000-014400 A JP 2001-037500 A Japanese Patent No. 2650159 Japanese Patent No. 3152927 Japanese Patent Laid-Open No. 8-211050 JP 2001-013147 A

Guidelines for diagnosis and treatment of sexually transmitted diseases, 49-56 (2008) Furuya Ryuzaburo, Nihon Lin, 67 (1), 129-135 (2009) Ishiguro T. et al. , Anal. Biochem. , 314, 77-86 (2003) Ishiguro T. et al. , Nucleic Acids Res. , 24, 4992-4997 (1996)

  Conventionally, methods for examining gonococcal infections that have been used in clinical practice include microscopic examination of gram-stained specimens of urethral secretions, examination by separation culture, and nucleic acid amplification examination. Although the microscopic inspection by Gram staining is a rapid inspection method, there is a problem that it is difficult to confirm the gonococcus when other bacteria are present. Tests using the culture method make it difficult to identify gonococci when mixed with other germs, are susceptible to heat, drying, and temperature changes, and are susceptible to death during sample transportation. It took several days to complete, so it was not suitable for rapid inspection. On the other hand, the nucleic acid amplification test is a method for amplifying and detecting genes (DNA and RNA) specific to Neisseria gonorrhoeae. As a method for amplifying DNA, PCR, SDA (Strand Displacement Amplification), etc. amplify RNA. As a method, a TMA method (Transcribation Mediated Amplification) (Patent Document 1) is used. The nucleic acid amplification test is extremely high in both sensitivity and specificity compared to the conventional test, and this test is becoming mainstream at present because a urine sample that is a non-invasive sample can be used.

  Test kits are commercially available for gonococcal testing using nucleic acid amplification. As an example, Cobas Amplicor STD-1 (Roche, using PCR method), BD Probetech ET (Becton Dickinson, using SDA method) Aptima Combo 2 (Gen Probe Co., Ltd., using TMA method). However, the gonococcus test using the kit has a problem of cross-reactivity with respect to Neisseria spp. Other than gonococcus and a problem that it takes a long time to complete the test (about 4 to 5 hours). In order for a doctor to make a diagnosis on the day of whether or not a patient visiting a medical institution is infected with Neisseria gonorrhoeae, a system capable of presenting the test results within at least two hours is preferable. Therefore, there is a need for a more rapid gonococcal inspection method.

  In addition to Neisseria meningitidis, which causes bacterial meningitis in humans, there are many non-pathogenic bacteria in Neisseria belonging to Neisseria gonorrhoeae. Exists. Some non-pathogenic Neisseria are commonly found in the human oral cavity as resident bacteria. Therefore, in the examination of gonococcus, particularly when examining the pharynx, it is necessary to have high specificity and high sensitivity to gonococcus.

  Therefore, an object of the present invention is to provide a method capable of obtaining a result with high sensitivity, high specificity, and speed in comparison with a conventional kit in a bacilli test using nucleic acid amplification.

  As a result of intensive studies in view of the above object, the present inventors have completed the present invention.

That is, the first aspect of the present invention is:
Using the first primer having a sequence homologous to a part of the specific base sequence in Neisseria gonorrhoeae 16S rRNA and the second primer having a sequence complementary to a part of the specific base sequence, the specific base A method for detecting gonococcal 16S rRNA in a sample by amplifying a polynucleotide comprising a sequence comprising:
The first primer is an oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence set forth in SEQ ID NO: 14;
The second primer is an oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence set forth in SEQ ID NO: 24.
The detection method.

The second aspect of the present invention is as follows.
The first primer is an oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the complementary sequence of the base sequence set forth in SEQ ID NO: 11;
The second primer is an oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the complementary sequence of the base sequence set forth in SEQ ID NO: 23.
The detection method according to the first aspect.

  The third aspect of the present invention is the first or second aspect, wherein at least one of the first and second primers has at least a promoter sequence of RNA polymerase added to the 5 ′ end thereof. The detection method described in 1.

The fourth aspect of the present invention is:
Amplification of a polynucleotide comprising a specific base sequence in Neisseria gonorrhoeae 16S rRNA,
(1) a step of synthesizing a cDNA complementary to a specific base sequence using aspergillus 16S rRNA as a template, using a second primer and an enzyme having RNA-dependent DNA polymerase activity;
(2) Degrading RNA-DNA double-stranded RNA with an enzyme having ribonuclease H (RNase H) activity (generation of single-stranded DNA),
(3) having a promoter sequence capable of transcribing a specific base sequence using the first primer and an enzyme having DNA-dependent DNA polymerase activity as a template or a sequence complementary to the specific base sequence. Generating double-stranded DNA;
(4) a step of generating an RNA transcript using the double-stranded DNA as a template by an enzyme having RNA polymerase activity; and (5) the RNA transcript is a template for cDNA synthesis in the step (1). A step of generating RNA transcripts in a chain,
It is the detection method as described in said 3rd aspect performed by.

  The fifth aspect of the present invention uses a probe that changes the fluorescence characteristics of the RNA transcript produced when a complementary double strand is formed with a part of the base sequence of the transcript. The fourth aspect, wherein the base sequence of the probe is a base sequence of an oligonucleotide that can be detected and hybridizes under stringent conditions with at least 10 consecutive bases in the base sequence set forth in SEQ ID NO: 30 or 31. The detection method described in 1.

The sixth aspect of the present invention is:
Prior to the step of synthesizing cDNA complementary to a specific base sequence using Aspergillus 16S rRNA as a template by the second primer and an enzyme having RNA-dependent DNA polymerase activity,
A cleaving oligonucleotide that overlaps the 5 ′ end side of the homologous region with the first primer in the specific base sequence and is complementary to a region adjacent to the 5 ′ end side from the overlapping site, and RNase H activity A step of cleaving the 5 ′ end of the specific base sequence with an enzyme having
The detection according to the fourth or fifth aspect, wherein the cleavage oligonucleotide is an oligonucleotide capable of hybridizing under stringent conditions with at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 7. Is the method.

Furthermore, the seventh aspect of the present invention is
An oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence set forth in SEQ ID NO: 14;
An oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence set forth in SEQ ID NO: 24;
Is a detection reagent for Neisseria gonorrhoeae 16S rRNA.

  Hereinafter, the present invention will be described in detail.

  In the detection method of the present invention, ribosomal RNA (rRNA) having a high content per cell (RNA copy number) was selected as a target in order to detect gonococci with high sensitivity. In addition, in order to increase the specificity for Neisseria gonorrhoeae, oligonucleotides (primers and probes) used in the detection method and detection reagent of the present invention were designed by searching for Neisseria gonorrhoeae rRNA sequences that are specific for Neisseria gonorrhoeae.

  Examples of samples in the present invention include food, drinking water, environmental water, feces, vomit, nasal cavity, pharynx, urethra, cervix, etc., gargle, gargle, blood, urine, other secretions, body fluids, tissue washings Can be given.

  In the present invention, the specific base sequence is an RNA sequence homologous to the base sequence from the 5 ′ end of the homologous region with the first primer to the 3 ′ end of the complementary region with the second primer in Neisseria gonorrhoeae 16S rRNA. Or DNA sequence. That is, in the present invention, a polynucleotide containing the specific base sequence or its complementary sequence is amplified.

  Examples of stringent conditions in the present invention include 50% (v / v) formamide, 0.1% bovine serum albumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate at 42 ° C. Examples include conditions in which a buffer (pH 6.5), 150 mM sodium chloride, and 75 mM sodium citrate are present, and RNA amplification conditions described in the examples of the present specification.

  Among the primers used in the detection method of the present invention, the first primer is at least 10 consecutive in the base sequence described in SEQ ID NO: 14 (complementary sequence of base sequence from 55th to 201st of GenBank No. AJ239304). The oligonucleotide may be any oligonucleotide that can hybridize with a base under stringent conditions, and the second primer is at least in the base sequence described in SEQ ID NO: 24 (base sequence from 387th to 448th base of GenBank No. AJ239304). Any oligonucleotide can be used as long as it can hybridize with 10 consecutive bases under stringent conditions. That is, the base sequence may be substituted, deleted, added, or modified as long as the base sequence can be sufficiently specifically and efficiently hybridized under the stringent conditions described above. The length of the primer can be arbitrarily set, but is preferably in the range of 10 to 50 bases.

  In the detection method of the present invention, detection is performed by amplifying a polynucleotide comprising a specific base sequence in Neisseria gonorrhoeae 16S rRNA. Examples of the method for amplifying the polynucleotide include RT-PCR method, RT-LAMP (Loop-mediated isometric Amplification) method, TMA method (Patent Document 1), NASBA (Nucleic Acid Sequence-Based Amplification) method (Patent Documents 4 and 4). 5) method and TRC (Transcribion Reverse transcription Concerted reaction) method (patent documents 2 and 3, non-patent document 3), which can be selected as appropriate. Among these, the TMA method, the NASBA method, or the TRC method, which is not affected by the contaminating DNA and can perform RNA amplification at a constant temperature, is preferable.

Amplification of RNA consisting of a specific base sequence in Neisseria gonorrhoeae 16S rRNA by TMA method, NASBA method or TRC method,
(1) a step of synthesizing a cDNA complementary to a specific base sequence using aspergillus 16S rRNA as a template, using a second primer and an enzyme having RNA-dependent DNA polymerase activity;
(2) a step of degrading RNA-DNA double-stranded RNA with an enzyme having RNase H activity (generation of single-stranded DNA),
(3) having a promoter sequence capable of transcribing a specific base sequence using the first primer and an enzyme having DNA-dependent DNA polymerase activity as a template or a sequence complementary to the specific base sequence. Generating double-stranded DNA;
(4) a step of generating an RNA transcript using the double-stranded DNA as a template by an enzyme having RNA polymerase activity; and (5) the RNA transcript is a template for cDNA synthesis in the step (1). A step of generating RNA transcripts in a chain,
To do.

  An enzyme having RNA-dependent DNA polymerase activity used in the step (1), an enzyme having RNase H activity used in the step (2), and a DNA-dependent DNA polymerase activity used in the step (3). Enzymes having different activities may be used, or enzymes having a plurality of activities may be used in combination, but it is preferable to use enzymes having all three activities. Examples of such preferred enzymes include AMV (Avian Myeloblastosis Virus) reverse transcriptase, MMLV (Molone Murine Leukemia Virus) reverse transcriptase, RAV (Rous Associated Virus) reverse transcriptase, and HIV (HumenVirum reverse transcriptase) Examples include retrovirus-derived reverse transcriptase that is usually used in the field of biology.

  As the enzyme having RNA polymerase activity used in the step (4), T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, etc. derived from bacterial phages widely used in the field of molecular biology can be used. By adding a promoter sequence corresponding to the enzyme having RNA polymerase activity used in the step (4) to the 5 ′ end side of the first or second primer, the specific base sequence in the koji mold 16S rRNA is added. The containing RNA is transcribed. A transcription initiation region that is known to affect transcription efficiency may be further added to the promoter sequence.

  The RNA amplification reaction comprising a specific base sequence in Neisseria gonorrhoeae 16S rRNA by the steps (1) to (5) can be performed at a constant temperature. The reaction temperature can be set at any temperature as long as the enzyme used in the above (1) to (4) is active. As an example, when the enzyme used in (1) to (3) is AMV reverse transcriptase and the enzyme used in (4) is T7 RNA polymerase, the reaction temperature is set in the range of 35 ° C to 65 ° C. What is necessary is just to set in the range of 40 to 50 degreeC.

For the detection of the RNA transcript generated in the step (4), a conventionally known nucleic acid detection method can be used,
(A) a method using electrophoresis or liquid chromatography,
(B) a hybridization method using a nucleic acid probe labeled with a detectable label;
(C) a method using a fluorescent substance-labeled probe designed to change fluorescence characteristics by hybridizing (complementary binding) with a partial base sequence of the transcription sequence,
Etc. However, the methods (A) and (B) are multi-step operations, and the amplification products are taken out of the system for analysis, and therefore the risk of scattering the amplification products to the environment causing secondary contamination. Is big. On the other hand, since the method (C) is a method that can solve the above-mentioned problems, it is preferable as a method for detecting an RNA transcript. Examples of the fluorescent substance labeled probe include a fluorescent labeled probe using FRET (fluorescence resonance energy transfer) and an intercalator labeled probe.

  The intercalator-labeled probe is intercalatoric via an appropriate linker to the phosphodiester part or base part of an oligonucleotide that can hybridize (complementary binding) to a part of the base sequence of the RNA transcript under stringent conditions. A probe that binds (labels) a fluorescent dye. When a complementary double strand is formed with an RNA transcript, the intercalating fluorescent dye portion intercalates with the complementary double strand portion, resulting in a change in fluorescence characteristics. Probe. By further including an intercalator-labeled probe in the reagent used in the measurement method of the present invention, the amplified RNA transcript is detected from the change in fluorescence characteristics while performing the RNA amplification reaction shown in (1) to (5) above. (Patent Document 2 and Non-Patent Document 3). As the intercalating fluorescent dye, conventionally known dyes can be used, and examples thereof include oxazole yellow, thiazole orange, ethidium bromide, hemicyanine, and derivatives thereof. Intercalating fluorescent dyes change the fluorescence intensity at a specific wavelength by intercalating into complementary double-stranded portions. For example, oxazole yellow significantly increases fluorescence at 510 nm (excitation wavelength: 490 nm) by intercalating into double-stranded DNA. When an intercalator-labeled probe is used in the detection method of the present invention, the hydroxyl group on the 3 ′ end side of the probe may be modified with an appropriate group to prevent nucleic acid elongation from the 3 ′ end side (Patent Document 6 and non-patent documents). Patent Document 4). Labeling of the intercalating fluorescent dye to the oligonucleotide can be performed by a conventionally known method (Patent Document 6 and Non-Patent Document 4), and a commercially available kit (for example, Label-ON Reagents (manufactured by Clontech)). May be used. Specifically, the base sequence of the intercalator-labeled probe used in the detection method of the present invention is SEQ ID NO: 30 (GenBank No. AJ239304 region 146 to 188) or SEQ ID NO: 31 (GenBank No. AJ239304). Any base sequence of an oligonucleotide that can hybridize under stringent conditions with at least 10 consecutive bases in the base sequence described in the 381st to 412th regions). That is, the base sequence may be substituted, deleted, added, or modified as long as the base sequence can be sufficiently specifically and efficiently hybridized under the stringent conditions described above. The length of the probe can be arbitrarily set, but is preferably in the range of 10 to 30 bases.

When a promoter sequence corresponding to the enzyme having RNA polymerase activity used in the step (4) is added to the 5 ′ end side of the first primer, before the step (1),
(0) Cleavage oligo that overlaps with the 5 ′ terminal side of the homologous region with the first primer in the specific base sequence in Neisseria gonorrhoeae 16S rRNA and that is complementary to the region adjacent to the 5 ′ terminal side from the overlapping site Cleaving the 5 ′ end of the specific nucleic acid sequence with a nucleotide and an enzyme having RNase H activity;
Is preferable. The DNA strand complementary to the promoter sequence of the first primer hybridized to the cDNA synthesized by the step (1) by cutting at the 5 ′ end site of the specific base sequence is By extending the 3 ′ terminal side of the cDNA in the step, it can be efficiently synthesized, resulting in the formation of a functional double-stranded DNA promoter structure. The enzyme having RNase H activity used in the step (0) may use the RNase H activity of the retrovirus-derived reverse transcriptase (for example, AMV reverse transcriptase) described above. It may be added. The hydroxyl group at the 3 ′ end of the cleavage oligonucleotide is preferably subjected to appropriate modification (for example, amination) in order to prevent the elongation reaction. Specifically, the oligonucleotide for cleavage used in the detection method of the present invention is at least 15 consecutive bases in the base sequence described in SEQ ID NO: 7 (the base sequence from the 41st to the 189th base sequence of GenBank No. AJ239304). Any oligonucleotide can be used as long as it can hybridize under stringent conditions. That is, the base sequence may be substituted, deleted, added, or modified as long as the base sequence can be sufficiently specifically and efficiently hybridized under the stringent conditions described above. The length of the oligonucleotide can be arbitrarily set, but is preferably in the range of 15 to 50 bases.

The detection reagent of the present invention comprises at least a first primer capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence set forth in SEQ ID NO: 14.
A second primer capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence set forth in SEQ ID NO: 24;
Should be included. As a preferred example of the first primer, an oligo capable of hybridizing under stringent conditions with at least 10 consecutive bases in the complementary sequence of the base sequence described in any of SEQ ID NOs: 8, 9, 10, 11, 13 And nucleotides. A preferred example of the second primer is an oligonucleotide that can hybridize under stringent conditions with at least 10 consecutive bases in the complementary sequence of the base sequence described in any of SEQ ID NOs: 15 to 23. In addition, either one of the first and second primers has at least a RNA polymerase promoter sequence (for example, a T7 promoter sequence comprising the sequence described in SEQ ID NO: 32) added to the 5 ′ end side thereof. preferable.

Furthermore, the detection reagent of the present invention includes
An intercalator-labeled probe in which an intercalator fluorescent dye is bound (labeled) to an oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence shown in SEQ ID NO: 30 or 31; and / or A cleavage oligonucleotide capable of hybridizing under stringent conditions with at least 15 consecutive bases in the base sequence set forth in SEQ ID NO: 7,
Is preferably included. Further, as a more preferable example of the cleavage nucleotide, it can hybridize under stringent conditions with at least 15 consecutive bases in the complementary sequence of the base sequence described in any of SEQ ID NOs: 1, 2, 3, 4, and 6. Examples include oligonucleotides. The nucleotide for cleavage needs to be a sequence that overlaps with the 5 ′ end side of the first primer and is complementary to a region adjacent to the 5 ′ side from the overlapping site. For example,
(I) When the first primer is an oligonucleotide having the base sequence described in SEQ ID NO: 8 (base sequence from GenBank No. AJ239304 from the 67th to the 86th base sequence), An oligonucleotide consisting of the described base sequence (complementary sequence of the base sequence from the 56th to the 75th base sequence of GenBank No. AJ239304);
(Ii) When the first primer is an oligonucleotide having the base sequence described in SEQ ID NO: 9 (base sequence from 55th to 74th base of GenBank No. AJ239304), An oligonucleotide consisting of the described base sequence (complementary sequence from the 41st to the 63rd base sequence of GenBank No. AJ239304),
(Iii) When the first primer is an oligonucleotide having the base sequence described in SEQ ID NO: 10 (base sequence from 94th to 117th base of GenBank No. AJ239304), An oligonucleotide consisting of the described base sequence (complementary sequence of the base sequence from the 78th to the 103rd base of GenBank No. AJ239304);
(Iv) When the first primer is an oligonucleotide having the base sequence described in SEQ ID NO: 11 (the 107th to 126th base sequences of GenBank No. AJ239304), An oligonucleotide consisting of the described base sequence (complementary sequence of base sequence from No. 93 to 115 of GenBank No. AJ239304),
(V) When the first primer is an oligonucleotide having the base sequence described in SEQ ID NO: 13 (base sequence from 182nd to 201st base of GenBank No. AJ239304), An oligonucleotide having the described base sequence (complementary sequence from base positions 170 to 189 of GenBank No. AJ239304) can be mentioned.

  In a sample containing Neisseria gonorrhoeae 16S rRNA, the oligonucleotide (primer / probe / cleaving oligonucleotide) contained in the detection reagent of the present invention, AMV reverse transcriptase, T7 RNA polymerase, buffer, magnesium salt, potassium salt, nucleoside-3 An amplification reagent containing phosphoric acid, ribonucleoside-triphosphate and dimethyl sulfoxide (DMSO) is added and reacted at a constant temperature of 35 to 65 ° C. (preferably 40 to 50 ° C.). Is measured over time, and amplification and measurement of Neisseria gonorrhoeae 16S rRNA in a sample can be performed within 30 minutes.

  The present invention uses a first primer having a sequence homologous to a part of a specific base sequence in Neisseria gonorrhoeae 16S rRNA, and a second primer having a sequence complementary to a part of the specific base sequence. , A method for detecting Neisseria gonorrhoeae 16S rRNA in a sample by amplifying a polynucleotide containing the specific base sequence, wherein the first primer is at least 10 consecutive in the base sequence set forth in SEQ ID NO: 14. An oligonucleotide capable of hybridizing under stringent conditions with a base, wherein the second primer is capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence of SEQ ID NO: 24 It is characterized by that. By using the first and second primers, gonococcal 16S rRNA contained in a trace amount in a sample can be rapidly and highly specific. Therefore, gonococci can be detected quickly and with high specificity regardless of the gene group or genotype.

  In the present invention, an intercalator label in which an intercalator fluorescent dye is bound (labeled) to an oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence described in SEQ ID NO: 30 or 31. Preferably it includes a probe. By adding the probe to the amplification system in advance, the specific base sequence in the Neisseria gonorrhoeae 16S rRNA is amplified by the first and second primers, and the amplified specific base sequence is changed by changing the fluorescence characteristics of the probe. It can be detected over time. Since the amplification reaction and the detection can be carried out in a sealed container without opening the cover after the sample and necessary components are added, it is also preferable in terms of preventing contamination.

  According to the present invention, the koji mold contained in the sample can be detected with high sensitivity, and the detection time is also quicker than that of the prior art. Therefore, it is possible to present a test result to a doctor more quickly than before, and it is considered that it contributes to prevention of infection spread and generation of resistant bacteria by administration of an appropriate drug.

The structure of the intercalating fluorescent dye-labeled nucleic acid probe produced in Example 2. B ¹ , B ² , B ³ and B ⁴ represent bases. In order to prevent an extension reaction from the hydroxyl group at the 3 ′ end, the hydroxyl group at the 3 ′ end is modified with glycolic acid.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by these Examples.

Example 1 Preparation of Standard RNA Neisseria gonorrhoeae 16S rRNA (hereinafter referred to as standard RNA) used in the following examples was prepared by the methods shown in the following (1) to (4).
(1) Using a RNA extract of Aspergillus oryzae (ATCC No. 19424), a double-stranded DNA of 16S rRNA base sequence region was prepared (Note that an SP6 promoter was added to the 5 ′ end side of the DNA) .
(2) The double-stranded DNA prepared in (1) was inserted into the multicloning site of the pUC19 plasmid according to a conventional method, and Escherichia coli JM109 strain was transformed.
(3) A plasmid was extracted from the transformed E. coli culture of (2), and the 3 ′ end of the inserted DNA was digested with restriction enzymes to prepare linear DNA.
(4) Using the DNA prepared in (3) as a template, in vitro transcription was performed using SP6 RNA polymerase. Subsequently, the double-stranded DNA was completely digested by DNase I treatment, and then RNA was purified. Standard RNA was prepared. The RNA was quantified by measuring the absorbance at 260 nm.

  The prepared standard RNA contains the base sequence of Aspergillus oryzae 16S rRNA and has a total length of about 1500 bases (8 bases derived from the SP6 promoter are added to the 5 'end of the RNA).

Example 2 Preparation of Oligonucleotide Probe (INAF Probe) Labeled with Intercalating Fluorescent Dye Oligonucleotide Probe Labeled with Intercalating Fluorescent Dye (hereinafter referred to as INAF Probe) It was prepared with. In particular,
Between the 8th cytosine and the 9th adenine from the 5 ′ end in the base sequence set forth in SEQ ID NO: 25 (complementary sequence of the 170th to 188th base sequences of GenBank No. AJ239304),
Between the 10th cytosine and the 11th cytosine from the 5 ′ end of the base sequence described in SEQ ID NO: 26 (complementary sequence of the base sequence from 176th to 195th of GenBank No. AJ239304),
7th thymine from the 5 ′ end to the 8th position in the base sequence shown in SEQ ID NO: 27 (complementary sequence from 381 to 402 of GenBank No. AJ239304 (where 389 is thymine, not cytosine)) During the guanine
Between the 4th cytosine and the 5th guanine from the 5 ′ end in the base sequence described in SEQ ID NO: 28 (complementary sequence of the 396th to 412th base sequences of GenBank No. AJ239304),
Between the 7th guanine and the 8th adenine from the 5 ′ end in the base sequence shown in SEQ ID NO: 29 (complementary sequence of the base sequence from the 146th to the 162nd base sequence of GenBank No. AJ239304),
An oxazole yellow-labeled nucleic acid probe was prepared by binding oxazole yellow to each of the phosphodiester moieties via a linker (FIG. 1).

Example 3 Investigation of Primer Set for Detection of Neisseria gonorrhoeae 16S rRNA (Part 1)
Measurement of standard RNA by the methods shown in (1) to (4) below using combinations (primer sets) of the first primer, the second primer, the INAF probe, and the cleavage oligonucleotide shown in Tables 1 and 2 To evaluate detection performance and specificity.
(1) The standard RNA prepared in Example 1 was prepared using an RNA diluent (10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA, 0.25 U / μL ribonuclease inhibitor, 5.0 mM DTT). ^It was diluted to ³ copies / 5 μL and used as an RNA sample.
(2) 20 μL of the reaction solution having the following composition was dispensed into a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, SSI), and 5 μL of the RNA sample was added thereto.

Composition of reaction solution: concentration is final concentration after addition of enzyme solution (in 30 μL) 60 mM Tris-HCl buffer (pH 8.6)
17 mM magnesium chloride 100 mM potassium chloride 1 mM DTT
0.25 mM dATP, dCTP, dGTP, dTTP each
Each 3.0 mM ATP, CTP, UTP, GTP
3.6 mM ITP
1.0 μM first primer (the primer has a T7 promoter sequence (SEQ ID NO: 32) added to the 5 ′ end side of the base sequence described in each SEQ ID NO)
1.0 μM second primer 25 nM INAF probe (prepared in Example 2)
Each 0.16 μM oligonucleotide for cleavage (the hydroxyl group at the 3 ′ end of the oligonucleotide is modified with an amino group)
6U ribonuclease inhibitor (Takara Bio Inc.)
13.0% DMSO
(3) After the above reaction solution was kept at 43 ° C. for 5 minutes, 5 μL of an enzyme solution having the following composition and kept at 43 ° C. for 2 minutes in advance was added.

Composition of enzyme solution: final concentration at the time of reaction (in 30 μL) 2.0% sorbitol 6.4 U AMV reverse transcriptase (Life Science)
142U T7 RNA polymerase (Invitrogen)
3.6 μg bovine serum albumin (4) Subsequently, using a fluorescence spectrophotometer with a temperature control function capable of directly measuring a PCR tube, the reaction solution was reacted at 43 ° C. and at the same time the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm) Measurement was performed for 30 minutes over time.

  The time when the enzyme is added is defined as positive when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value of the predetermined time by the fluorescence intensity ratio of the background) exceeds 1.2. Tables 1 and 2 show the results obtained from the detection time. “Nega” in the table refers to a reaction obtained by adding distilled water for injection instead of the target nucleic acid. N. in the table. D. Means a sample having a fluorescence intensity ratio of less than 1.2 (negative determination) 30 minutes after the enzyme was added. “Determination” in the table is a comprehensive judgment from the detection time of standard RNA, the measurement result of Nega, the fluorescence intensity ratio, etc., and “○”, “△”, “×” in order from the best combination. It is described.

表１および２の結果より、
切断用オリゴヌクレオチドとして、配列番号７に記載の塩基配列中の少なくとも連続する１５塩基とストリンジェントな条件でハイブリダイズ可能なオリゴヌクレオチドの一例である、
配列番号１に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の５６番目から７５番目までの塩基配列の相補配列）、
配列番号２に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の４１番目から６３番目までの塩基配列の相補配列）、
配列番号３に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の７８番目から１０３番目までの塩基配列の相補配列）、
配列番号４に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の９３番目から１１５番目までの塩基配列の相補配列）、
配列番号６に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の１７０番目から１８９番目までの塩基配列の相補配列）、のいずれかからなるオリゴヌクレオチドが、
第一のプライマーとして、配列番号１４に記載の塩基配列中の少なくとも連続する１０塩基とストリンジェントな条件でハイブリダイズ可能なオリゴヌクレオチドの一例である、配列番号８に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の６７番目から８６番目までの塩基配列）、
配列番号９に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の５５番目から７４番目までの塩基配列）、
配列番号１０に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の９４番目から１１７番目までの塩基配列）、
配列番号１１に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の１０７番目から１２６番目までの塩基配列）、
配列番号１３に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の１８２番目から２０１番目までの塩基配列）、のいずれかからなるオリゴヌクレオチドが、
第二のプライマーとして、配列番号２４に記載の塩基配列中の少なくとも連続する１０塩基とストリンジェントな条件でハイブリダイズ可能なオリゴヌクレオチドの一例である、
配列番号１５に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の３８７番目から４０４番目までの塩基配列の相補配列）、
配列番号１６に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の４０２番目から４１８番目までの塩基配列の相補配列）、
配列番号１７に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の４０３番目から４１９番目までの塩基配列の相補配列）、
配列番号１８に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の４０６番目から４２５番目までの塩基配列の相補配列）、
配列番号１９に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の４１２番目から４３５番目までの塩基配列の相補配列）、
配列番号２０に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の４２６番目から４４８番目までの塩基配列の相補配列）、のいずれかからなるオリゴヌクレオチドが、
ＩＮＡＦプローブを構成するオリゴヌクレオチドとして、
配列番号３０または３１に記載の塩基配列中の少なくとも連続する１０塩基とストリンジェントな条件でハイブリダイズ可能なオリゴヌクレオチドの一例である、
配列番号２５に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の１７０番目から１８８番目までの塩基配列の相補配列）、
配列番号２７に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の３８１番目から４０２番目までの塩基配列（ただし３８９番目はシトシンではなくチミン）の相補配列）、
配列番号２８に記載の塩基配列（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の３９６番目から４１２番目までの塩基配列の相補配列）、のいずれかからなるオリゴヌクレオチドが、
それぞれ使用できることが分かった。

From the results in Tables 1 and 2,
As an oligonucleotide for cleavage, it is an example of an oligonucleotide that can hybridize under stringent conditions with at least 15 consecutive bases in the base sequence set forth in SEQ ID NO: 7.
The base sequence described in SEQ ID NO: 1 (complementary sequence of the base sequence from the 56th to the 75th base sequence of GenBank No. AJ239304),
The base sequence described in SEQ ID NO: 2 (complementary sequence of the base sequence from the 41st to the 63rd base sequence of GenBank No. AJ239304),
The base sequence described in SEQ ID NO: 3 (complementary sequence of the base sequence from the 78th to the 103rd base of GenBank No. AJ239304),
The base sequence described in SEQ ID NO: 4 (complementary sequence of the base sequence from the 93rd position to the 115th position of GenBank No. AJ239304),
An oligonucleotide consisting of any of the nucleotide sequences set forth in SEQ ID NO: 6 (complementary sequences of the 170th to 189th nucleotide sequences of GenBank No. AJ239304),
As a first primer, the base sequence described in SEQ ID NO: 8 (GenBank No. 1), which is an example of an oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence described in SEQ ID NO: 14. 67th to 86th base sequences of AJ239304)
The base sequence described in SEQ ID NO: 9 (base sequence from GenBank No. AJ239304 from the 55th position to the 74th position),
The base sequence described in SEQ ID NO: 10 (base sequence from GenBank No. AJ239304 from the 94th position to the 117th position),
The base sequence described in SEQ ID NO: 11 (the base sequence from the 107th position to the 126th position of GenBank No. AJ239304),
An oligonucleotide consisting of any one of the base sequences set forth in SEQ ID NO: 13 (base sequences from 182nd to 201st of GenBank No. AJ239304),
The second primer is an example of an oligonucleotide that can hybridize under stringent conditions with at least 10 consecutive bases in the base sequence set forth in SEQ ID NO: 24.
The base sequence described in SEQ ID NO: 15 (complementary sequence of the base sequence from the 387th to the 404th base of GenBank No. AJ239304),
The base sequence described in SEQ ID NO: 16 (complementary sequence of base sequence from position 402 to position 418 of GenBank No. AJ239304),
The base sequence described in SEQ ID NO: 17 (complementary sequence of base sequence from 403rd to 419th of GenBank No. AJ239304),
The base sequence described in SEQ ID NO: 18 (complementary sequence of the base sequence from 406th to 425th of GenBank No. AJ239304),
The base sequence described in SEQ ID NO: 19 (complementary sequence of the base sequence from 412 to 435 of GenBank No. AJ239304),
An oligonucleotide consisting of any of the nucleotide sequences set forth in SEQ ID NO: 20 (complementary sequences of nucleotide sequences 426 to 448 of GenBank No. AJ239304),
As an oligonucleotide constituting the INAF probe,
It is an example of an oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence set forth in SEQ ID NO: 30 or 31.
The nucleotide sequence set forth in SEQ ID NO: 25 (complementary sequence of nucleotide sequence 170 to 188 of GenBank No. AJ239304),
The base sequence described in SEQ ID NO: 27 (complementary sequence of the 381st to 402nd base sequences of GenBank No. AJ239304 (where 389th is not cytosine but thymine)
An oligonucleotide consisting of any of the nucleotide sequences set forth in SEQ ID NO: 28 (complementary sequences of the 396th to 412th nucleotide sequences of GenBank No. AJ239304),
It turns out that each can be used.

Example 4 Investigation of Primer Set for Detection of Neisseria gonorrhoeae 16S rRNA (Part 2)
As a result of Example 3, standard RNA in a low concentration region was measured for the following combinations judged to have particularly good performance among the combinations of oligonucleotides judged as “◯”. Note experimental method, 10 ² copies / 5 [mu] L as RNA sample, 300 copies / 5 [mu] L, another with 10 ³ copies / 5 [mu] L, the same as in Example 3.
(Combination A)
Oligonucleotide for cleavage: SEQ ID NO: 1, first primer: SEQ ID NO: 8
Second primer: SEQ ID NO: 15, IAF probe: SEQ ID NO: 25
(Combination M)
Cleavage oligonucleotide: SEQ ID NO: 2, first primer: SEQ ID NO: 9
Second primer: SEQ ID NO: 15, IAF probe: SEQ ID NO: 25
(Combination AK)
Oligonucleotide for cleavage: SEQ ID NO: 4, first primer: SEQ ID NO: 11
Second primer: SEQ ID NO: 15, IAF probe: SEQ ID NO: 25
(Combination AM)
Oligonucleotide for cleavage: SEQ ID NO: 4, first primer: SEQ ID NO: 11
Second primer: SEQ ID NO: 17, IAF probe: SEQ ID NO: 25
(Combination BK)
Cleavage oligonucleotide: SEQ ID NO: 6, first primer: SEQ ID NO: 13
Second primer: SEQ ID NO: 17, INAF probe: SEQ ID NO: 27
(Combination BS)
Cleavage oligonucleotide: SEQ ID NO: 6, first primer: SEQ ID NO: 13
Second primer: SEQ ID NO: 19, IAF probe: SEQ ID NO: 28
The results are shown in Table 3. Of the six combinations examined in this example, the combination A / AK / AM / BK / BS was found to be particularly preferable for detecting gonococcal 16S rRNA at low concentrations.

実施例５ 淋菌１６Ｓ ｒＲＮＡ検出用プライマーセットの検討（その３）
実施例４の結果、低濃度の淋菌１６Ｓ ｒＲＮＡ検出に好ましいとされた５つの組み合わせ（組み合わせＡ／ＡＫ／ＡＭ／ＢＫ／ＢＳ）について、反応特異性（交差反応性）を検討した。なお実験方法は、ＲＮＡ試料として下記に示す淋菌以外のナイセリア属菌培養物のＲＮＡ抽出物を用いた他は、実施例３と同様である。また培養物からのＲＮＡ抽出には、ＲＮｅａｓｙ ｍｉｎｉ ｋｉｔ（キアゲン社）を使用した。

Example 5 Investigation of primer set for detecting Neisseria gonorrhoeae 16S rRNA (part 3)
As a result of Example 4, the reaction specificity (cross-reactivity) was examined for five combinations (combination A / AK / AM / BK / BS) that were considered preferable for detecting low-concentration Neisseria gonorrhoeae 16S rRNA. The experimental method was the same as in Example 3 except that the RNA extract of the Neisseria spp. Culture other than Neisseria gonorrhoeae shown below was used as the RNA sample. For RNA extraction from the culture, RNeasy mini kit (Qiagen) was used.

Neisseria elongata (ATCC No. 25295)
Neisseria sicca (ATCC No. 29256)
Neisseria cinerea (ATCC No. 14685)
Neisseria lactamica (ATCC No. 23970)
Neisseria subflava (ATCC No. 49275)
Neisseria flava (ATCC No. 14221)
Neisseria mucosa (ATCC No. 25999)
Neisseria flavescens (ATCC No. 13120)
The results are shown in Table 4. Of the five combinations examined in this example, the combination AK / AM / BS was found to have a particularly high reaction specificity (particularly low cross-reactivity) for Neisseria gonorrhoeae 16S rRNA.

また、淋菌１６Ｓ ｒＲＮＡに対する反応特異性が特に高かった３つの組み合わせ（組み合わせＡＫ／ＡＭ／ＢＳ）について、各反応液中のＲＮＡ増幅産物の有無をアガロース電気泳動にて確認した。結果を表５に示す。表５において、電気泳動における＋の数は、増幅産物に由来するバンドの濃さの程度を示したものであり、＋の数が多いほど増幅産物量が多いことを表している。−は増幅産物が生成しなかったことを表している。前記３つの組み合わせのうち、組み合わせＡＭがＲＮＡ増幅段階での特異性が最も高いことが判明した。

In addition, for the three combinations (combination AK / AM / BS) that had a particularly high reaction specificity for Neisseria gonorrhoeae 16S rRNA, the presence or absence of RNA amplification products in each reaction solution was confirmed by agarose electrophoresis. The results are shown in Table 5. In Table 5, the number of + in electrophoresis indicates the degree of darkness of the band derived from the amplification product, and the greater the number of +, the greater the amount of amplification product. -Indicates that no amplification product was produced. Of the three combinations, the combination AM was found to have the highest specificity at the RNA amplification stage.

実施例６ 淋菌１６Ｓ ｒＲＮＡ検出用プライマーセットの検討（その４）
実施例５で選定したオリゴヌクレオチドの組み合わせ（組み合わせＡＭ）のうち、第二のプライマーを、配列番号２１（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の４０３番目から４１８番目までの塩基配列の相補配列）、配列番号２２（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の３９５番目から４１４番目までの塩基配列の相補配列）または配列番号２３（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の３９５番目から４０９番目までの塩基配列の相補配列）からなるオリゴヌクレオチドに変更して、Ｎｅｉｓｓｅｒｉａ ｃｉｎｅｒｅａ（ＡＴＣＣ Ｎｏ．１４６８５）存在下での淋菌１６Ｓ ｒＲＮＡ検出を検討した。なお実験方法は、ＲＮＡ試料として、５０ｃｆｕ（ｃｏｌｏｎｙ ｆｏｒｍｉｎｇ ｕｎｉｔ）／ｔｅｓｔの淋菌ＲＮＡ抽出物と１０^２ｃｆｕ／ｔｅｓｔから１０^７ｃｆｕ／ｔｅｓｔのＮｅｉｓｓｅｒｉａ ｃｉｎｅｒｅａ（ＡＴＣＣ Ｎｏ．１４６８５）ＲＮＡ抽出物との混合物を用いた他は、実施例３と同様である。また菌からのＲＮＡ抽出には、ＲＮｅａｓｙ ｍｉｎｉ ｋｉｔ（キアゲン社）を使用した。

Example 6 Investigation of Primer Set for Detection of Neisseria gonorrhoeae 16S rRNA (Part 4)
Among the oligonucleotide combinations (combination AM) selected in Example 5, the second primer was SEQ ID NO: 21 (complementary sequence of base sequences from 403th to 418th positions of GenBank No. AJ239304), SEQ ID NO: 22 ( A nucleotide sequence complementary to the 395th to 414th base sequences of GenBank No. AJ239304) or an oligonucleotide consisting of SEQ ID NO: 23 (complementary sequence of the 395th to 409th base sequences of GenBank No. AJ239304), Detection of Neisseria gonorrhoeae 16S rRNA in the presence of Neisseria cinerea (ATCC No. 14685) was examined. In the experimental method, a mixture of a bacillus RNA extract of 50 cfu (colony forming unit) / test and a Neisseria cinerea (ATCC No. 14685) RNA extract of 10 ² cfu / test to 10 ⁷ cfu / test is used as an RNA sample. Other than using, it is the same as that of Example 3. In addition, RNeasy mini kit (Qiagen) was used for RNA extraction from bacteria.

  The results are shown in Table 6. It was found that by changing the second primer from SEQ ID NO: 17 (combination AM) to SEQ ID NO: 23, gonococcal 16S rRNA can be detected even in the presence of a high concentration of Neisseria cinerea (ATCC No. 14685).

実施例７ 淋菌１６Ｓ ｒＲＮＡ検出用プライマーセットの検討（その５）
実施例６で選定したオリゴヌクレオチドの組み合わせ（切断用オリゴヌクレオチド：配列番号４、第一のプライマー：配列番号１１、第二のプライマー：配列番号２３、ＩＮＡＦプローブ：配列番号２５）のうち、ＩＮＡＦプローブを配列番号２８（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の３９６番目から４１２番目までの塩基配列の相補配列）または配列番号２９（ＧｅｎＢａｎｋ Ｎｏ．ＡＪ２３９３０４の１４６番目から１６２番目までの塩基配列の相補配列）からなるプローブに変更して、髄膜炎菌（Ｎｅｉｓｓｅｒｉａ ｍｅｎｉｎｇｉｔｉｄｉｓ）に対する交差反応性を検討した。なお実験方法は、ＲＮＡ試料として下記に示す髄膜炎菌培養物のＲＮＡ抽出物を用いた他は、実施例３と同様である。また培養物からのＲＮＡ抽出には、ＲＮｅａｓｙ ｍｉｎｉ ｋｉｔ（キアゲン社）を使用した。

Example 7 Examination of primer set for detecting Neisseria gonorrhoeae 16S rRNA (part 5)
Of the combinations of oligonucleotides selected in Example 6 (cleaving oligonucleotide: SEQ ID NO: 4, first primer: SEQ ID NO: 11, second primer: SEQ ID NO: 23, INAF probe: SEQ ID NO: 25), the INAF probe To a probe consisting of SEQ ID NO: 28 (complementary sequence of the 396th to 412th base sequences of GenBank No. AJ239304) or SEQ ID NO: 29 (complementary sequence of the 146th to 162nd base sequences of GenBank No. AJ239304) Modified to examine cross-reactivity to Neisseria meningitidis. The experimental method was the same as in Example 3 except that the RNA extract of the meningococcal culture shown below was used as the RNA sample. For RNA extraction from the culture, RNeasy mini kit (Qiagen) was used.

Neisseria meningitidis (serotype A) (ATCC No. 13077)
Neisseria meningitidis (serotype B) (ATCC No. BAA-335)
Neisseria meningitidis (serotype C) (ATCC No. 13102)
Neisseria meningitidis (serotype D) (ATCC No. 13113)
Neisseria meningitidis (serotype Y) (ATCC No. 35561)
Neisseria meningitidis (serotype W135) (ATCC No. 35559)
The results are shown in Table 7. It was found that there was no cross-reactivity to Neisseria meningitidis when using the INF probe consisting of the nucleotide sequence set forth in SEQ ID NO: 29.

実施例３から７までの検討で得られた、淋菌１６Ｓ ｒＲＮＡを増幅検出するのに最適なオリゴヌクレオチドの組み合わせを表８に示す。

Table 8 shows the optimal oligonucleotide combinations for amplifying and detecting Neisseria gonorrhoeae 16S rRNA obtained in the examination of Examples 3 to 7.

実施例８ 選定したプライマーセットを用いた淋菌の検出
表８に記載のオリゴヌクレオチドの組み合わせ（プライマーセット）を用い、下記（１）から（３）に示す方法で淋菌の検出を行なった。
（１）トリプチケースソイ（Ｔｒｙｐｔｉｃａｓｅ Ｓｏｙ）培地（ベクトンディッキンソン社）を用い、５％ＣＯ_２環境下で液体培養した淋菌（ＡＴＣＣ Ｎｏ．１９４２４）をＰＢＳで段階希釈し、希釈系列を調製した。淋菌の段階希釈液は、チョコレートＩＩ寒天培地（ベクトンディッキンソン社）に接種し、コロニー数をカウントすることで濃度を決定した。
（２）（１）で調製した淋菌希釈系列を、ＥＸＴＲＡＧＥＮ ＩＩ（東ソー社）で抽出し、ＲＮＡ抽出物を調製した。
（３）オリゴヌクレオチドの組み合わせとして表８の組み合わせを、ＲＮＡ試料として（２）で調製したＲＮＡ抽出物を用いた他は、実施例３に記載と同様な方法で測定を行なった。

Example 8 Detection of Neisseria gonorrhoeae using the selected primer set Using the oligonucleotide combinations (primer sets) listed in Table 8, Neisseria gonorrhoeae was detected by the methods shown in (1) to (3) below.
(1) Using a Trypticase Soy medium (Becton Dickinson), koji molds (ATCC No. 19424) cultured in a liquid in a 5% CO ₂ environment were serially diluted with PBS to prepare a dilution series. The serial dilution of Aspergillus was inoculated into Chocolate II agar medium (Becton Dickinson), and the concentration was determined by counting the number of colonies.
(2) The gonococcal dilution series prepared in (1) was extracted with EXTRAGEN II (Tosoh Corporation) to prepare an RNA extract.
(3) Measurement was performed in the same manner as described in Example 3, except that the combinations shown in Table 8 were used as the oligonucleotide combinations and the RNA extract prepared in (2) was used as the RNA sample.

  The results are shown in Table 9. It was found that the detection was possible up to 0.03 cfu / test of Aspergillus and the detection time was 20 minutes or less, and the measurement was extremely quick and highly sensitive.

Claims (7)

Using the first primer having a sequence homologous to a part of the specific base sequence in Neisseria gonorrhoeae 16S rRNA and the second primer having a sequence complementary to a part of the specific base sequence, the specific base A method for detecting gonococcal 16S rRNA in a sample by amplifying a polynucleotide comprising a sequence comprising:
The first primer is an oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence set forth in SEQ ID NO: 14;
The second primer is an oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence set forth in SEQ ID NO: 24.
The detection method. The first primer is an oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the complementary sequence of the base sequence set forth in SEQ ID NO: 11;
The second primer is an oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the complementary sequence of the base sequence set forth in SEQ ID NO: 23.
The detection method according to claim 1. The detection method according to claim 1 or 2, wherein at least one of the first and second primers has at least an RNA polymerase promoter sequence added to the 5 'end. Amplification of a polynucleotide comprising a specific base sequence in Neisseria gonorrhoeae 16S rRNA,
(1) a step of synthesizing a cDNA complementary to a specific base sequence using aspergillus 16S rRNA as a template, using a second primer and an enzyme having RNA-dependent DNA polymerase activity;
(2) Degrading RNA-DNA double-stranded RNA with an enzyme having ribonuclease H (RNase H) activity (generation of single-stranded DNA),
(3) having a promoter sequence capable of transcribing a specific base sequence using the first primer and an enzyme having DNA-dependent DNA polymerase activity as a template or a sequence complementary to the specific base sequence. Generating double-stranded DNA;
(4) a step of generating an RNA transcript using the double-stranded DNA as a template by an enzyme having RNA polymerase activity; and (5) the RNA transcript is a template for cDNA synthesis in the step (1). A step of generating RNA transcripts in a chain,
The detection method according to claim 3, wherein When the generated RNA transcript is formed into a complementary double strand with a part of the base sequence of the transcript, it is detected using a probe whose fluorescence characteristics change compared to before the formation, and the base sequence of the probe is The detection method according to claim 4, which is a base sequence of an oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence shown in SEQ ID NO: 30 or 31. Prior to the step of synthesizing cDNA complementary to a specific base sequence using Aspergillus 16S rRNA as a template by the second primer and an enzyme having RNA-dependent DNA polymerase activity,
A cleaving oligonucleotide that overlaps the 5 ′ end side of the homologous region with the first primer in the specific base sequence and is complementary to a region adjacent to the 5 ′ end side from the overlapping site, and RNase H activity A step of cleaving the 5 ′ end of the specific base sequence with an enzyme having
The detection method according to claim 4 or 5, wherein the cleaving oligonucleotide is an oligonucleotide that can hybridize under stringent conditions with at least 15 consecutive bases in the base sequence shown in SEQ ID NO: 7. An oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence set forth in SEQ ID NO: 14;
An oligonucleotide capable of hybridizing under stringent conditions with at least 10 consecutive bases in the base sequence set forth in SEQ ID NO: 24;
A detection reagent for Neisseria gonorrhoeae 16S rRNA, comprising at least

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